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.

Newly formed dust within the circumstellar environment of SN Ia-CSM 2018evt.

Dust associated with various stellar sources in galaxies at all cosmic epochs remains a controversial topic, particularly whether supernovae play an important role in dust production. We report evidence of dust formation in the cold, dense shell behind the ejecta-circumstellar medium (CSM) interaction in the Type Ia-CSM supernova (SN) 2018evt three years after the explosion, characterized by a rise in mid-infrared emission accompanied by an accelerated decline in the optical radiation of the SN. Such a dust-formation picture is also corroborated by the concurrent evolution of the profiles of the Hα emission line. Our model suggests enhanced CSM dust concentration at increasing distances from the SN as compared to what can be expected from the density profile of the mass loss from a steady stellar wind. By the time of the last mid-infrared observations at day +1,041, a total amount of 1.2 ± 0.2 × 10-2 M⊙ of new dust has been formed by SN 2018evt, making SN 2018evt one of the most prolific dust factories among supernovae with evidence of dust formation. The unprecedented witness of the intense production procedure of dust may shed light on the perceptions of dust formation in cosmic history.

Phase 1 trial evaluating safety and pharmacokinetics of HIV-1 broadly neutralizing mAbs 10E8VLS and VRC07-523LS.

BACKGROUNDBroadly neutralizing monoclonal antibodies (bNAbs) represent a promising strategy for HIV-1 immunoprophylaxis and treatment. 10E8VLS and VRC07-523LS are bNAbs that target the highly conserved membrane-proximal external region (MPER) and the CD4-binding site of the HIV-1 viral envelope glycoprotein, respectively.METHODSIn this phase 1, open-label trial, we evaluated the safety and pharmacokinetics of 5 mg/kg 10E8VLS administered alone, or concurrently with 5 mg/kg VRC07-523LS, via s.c. injection to healthy non-HIV-infected individuals.RESULTSEight participants received either 10E8VLS alone (n = 6) or 10E8VLS and VRC07-523LS in combination (n = 2). Five (n = 5 of 8, 62.5%) participants who received 10E8VLS experienced moderate local reactogenicity, and 1 participant (n = 1/8, 12.5%) experienced severe local reactogenicity. Further trial enrollment was stopped, and no participant received repeat dosing. All local reactogenicity resolved without sequelae. 10E8VLS retained its neutralizing capacity, and no functional anti-drug antibodies were detected; however, a serum t1/2 of 8.1 days was shorter than expected. Therefore, the trial was voluntarily stopped per sponsor decision (Vaccine Research Center, National Institute of Allergy and Infectious Diseases [NIAID], NIH). Mechanistic studies performed to investigate the underlying reason for the reactogenicity suggest that multiple mechanisms may have contributed, including antibody aggregation and upregulation of local inflammatory markers.CONCLUSION10E8VLS resulted in unexpected reactogenicity and a shorter t1/2 in comparison with previously tested bNAbs. These studies may facilitate identification of nonreactogenic second-generation MPER-targeting bNAbs, which could be an effective strategy for HIV-1 immunoprophylaxis and treatment.TRIAL REGISTRATIONClinicaltrials.gov, accession no. NCT03565315.FUNDINGDivision of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH.

Correlation of handgrip strength with quality of life-adjusted pulmonary function in adults.

BACKGROUND: Handgrip strength (HGS) is acknowledged as a key indicator of overall physical fitness and is associated with various health outcomes. OBJECTIVES: This research investigates the correlation between HGS and quality of life (QoL), focusing on its relation to pulmonary function in the general adult population. METHOD: The study involved 19,402 participants aged 40 and above, spanning from 2014 to 2019, who underwent pulmonary function and HGS tests. Participants were categorized based on lung function, and regression analyses were employed to examine the relationship between HGS and QoL, with adjustments made for lung function. RESULTS: The average age of the cohort was 58.2 years, comprising 44.6% males and 41.2% smokers. Out of the 18,708 participants who completed the European Quality of Life Scale-Five Dimensions (EQ-5D-3L) assessment, higher severity levels in mobility, self-care, usual activities, pain or discomfort, and anxiety or depression were linked to lower HGS in both sexes. Additionally, among the 3,723 participants who completed the Health-related Quality of Life Instrument with 8 Items (HINT-8) assessment, higher severity levels in pain, work, and depression were associated with lower HGS in men. In women, higher severity levels in climbing stairs, pain, vitality, and work correlated with lower HGS. CONCLUSIONS: As problems indicated by EQ-5D worsened, there was a consistent decrease in handgrip strength (HGS) across both genders. The HINT-8 assessment further revealed that increased severity in pain and work-related issues led to reduced HGS in both men and women. This study highlights the relationship between HGS and Quality of Life (QoL), taking lung function into consideration, and underscores the importance of HGS as a potential marker of physical health and fitness.

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.

CYP2D6-guided opioid therapy for adults with cancer pain: A randomized implementation clinical trial.

INTRODUCTION: The CYP2D6 enzyme metabolizes opioids commonly prescribed for cancer-related pain, and CYP2D6 polymorphisms may contribute to variability in opioid response. We evaluated the feasibility of implementing CYP2D6-guided opioid prescribing for patients with cancer and reported pilot outcome data. METHODS: Adult patients from two cancer centers were prospectively enrolled into a hybrid implementation-effectiveness clinical trial and randomized to CYP2D6-genotype-guided opioid selection, with clinical recommendations, or usual care. Implementation metrics, including provider response, medication changes consistent with recommendations, and patient-reported pain and symptom scores at baseline and up to 8 weeks, were assessed. RESULTS: Most (87/114, 76%) patients approached for the study agreed to participate. Of 85 patients randomized, 71% were prescribed oxycodone at baseline. The median (range) time to receive CYP2D6 test results was 10 (3-37) days; 24% of patients had physicians acknowledge genotype results in a clinic note. Among patients with CYP2D6-genotype-guided recommendations to change therapy (n = 11), 18% had a change congruent with recommendations. Among patients who completed baseline and follow-up questionnaires (n = 48), there was no difference in change in mean composite pain score (-1.01 ± 2.1 vs. -0.41 ± 2.5; p = 0.19) or symptom severity at last follow-up (3.96 ± 2.18 vs. 3.47 ± 1.78; p = 0.63) between the usual care arm (n = 26) and genotype-guided arm (n = 22), respectively. CONCLUSION: Our study revealed high acceptance of pharmacogenetic testing as part of a clinical trial among patients with cancer pain. However, provider response to genotype-guided recommendations was low, impacting assessment of pain-related outcomes. Addressing barriers to utility of pharmacogenetics results and clinical recommendations will be critical for implementation success.

Food environment index and preterm birth rate in the counties of the United States.

OBJECTIVES: To determine the relationship between Food Environment Index (FEI) and Preterm Birth (PTB) rate at the county level of the United States of America (USA) (primary), while evaluating the interaction of multiple factors within a framework of sociodemographic, maternal health, maternal behavioral, and environmental factors. METHODS: This is a population-based retrospective cohort ecological study from 2015-2018. The study compares the characteristics of the population of the counties of the USA. All counties with complete data on their PTB rate and the independent variables were included in the study. Independent variables with greater than 20% missing data were excluded from the study. Purposive sampling technique was applied. A total of 2983/3142 counties were included in the study. RESULTS: The median PTB rate of all counties was 9.90%. The highest PTB rate (23.3%) was in Tallapoosa County, Alabama and the lowest (3.4%) in San Juan County, Washington State. After adjusting for variables, PTB rate had a significant association with FEI (coefficient of correlation - 0.36, p < 0.01, 95% CI - 0.19 to - 0.04). Increase in the rate of unemployment, African American race, adult smoking, obesity, uninsured rate, sexually transmitted diseases (STD), high school education and air pollution was associated with an increase in PTB rate, while an increase in FEI and alcohol abuse rates was associated with a decrease in PTB rate. CONCLUSIONS: FEI can predict the PTB rate in USA counties after adjusting for sociodemographic, health, behavioral and environmental factors. Future studies are needed to confirm these associations and consider them when making policies to reduce PTBs.

Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization.

The third variable (V3) loop on the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein trimer is indispensable for virus cell entry. Conformational masking of V3 within the trimer allows efficient neutralization via V3 only by rare, broadly neutralizing glycan-dependent antibodies targeting the closed prefusion trimer but not by abundant antibodies that access the V3 crown on open trimers after CD4 attachment. Here, we report on a distinct category of V3-specific inhibitors based on designed ankyrin repeat protein (DARPin) technology that reinstitute the CD4-bound state as a key neutralization target with up to >90% breadth. Broadly neutralizing DARPins (bnDs) bound V3 solely on open envelope and recognized a four-turn amphipathic α-helix in the carboxy-terminal half of V3 (amino acids 314-324), which we termed 'αV3C'. The bnD contact surface on αV3C was as conserved as the CD4 binding site. Molecular dynamics and escape mutation analyses underscored the functional relevance of αV3C, highlighting the potential of αV3C-based inhibitors and, more generally, of postattachment inhibition of HIV-1.

Improved pharmacokinetics of HIV-neutralizing VRC01-class antibodies achieved by reduction of net positive charge on variable domain.

The amino-acid composition of the immunoglobulin variable region has been observed to impact antibody pharmacokinetics (PK). Here, we sought to improve the PK of the broad HIV-1-neutralizing VRC01-class antibodies, VRC07-523LS and N6LS, by reducing the net positive charge in their variable domains. We used a structure-guided approach to generate a panel of antibody variants incorporating select Arg or Lys substituted to Asp, Gln, Glu, or Ser. The engineered variants exhibited reduced affinity to heparin, reduced polyreactivity, and improved PK in human FcRn-transgenic mice. One variant, VRC07-523LS.v34, with three charge substitutions, had an observed in vivo half-life and an estimated human half-life of 10.8 and 60 days, respectively (versus 5.4 and 38 days for VRC07-523LS) and retained functionality, neutralizing 92% of a 208-strain panel at a geometric mean IC80 <1 µg/mL. Another variant, N6LS.C49, with two charge substitutions, had an observed in vivo half-life and an estimated human half-life of 14.5 and 80 days (versus 9.0 and 44 days for N6LS) and neutralized ~80% of 208 strains at a geometric mean IC80 <1 µg/mL. Since Arg and Lys residues are prevalent in human antibodies, we propose substitution of select Arg or Lys with Asp, Gln, Glu, or Ser in the framework region as a general means to improve PK of therapeutic antibodies.

Evaluating the Use of CT-Derived Lung Volumes in Donor-Recipient Lung Size Matching for Lung Transplantation in Patients With Interstitial Lung Disease and/or Idiopathic Pulmonary Fibrosis.

PURPOSE: This study aims to assess the efficacy of current measurement strategies for lung sizing and the feasibility of future use of computed tomography (CT)-derived lung volumes to predict a donor-recipient lung size match during bilateral lung transplants. METHODS: We reviewed the data of 62 patients who underwent bilateral lung transplantation for interstitial lung disease and/or idiopathic pulmonary fibrosis from 2018 to 2019. Data for recipients was retrieved from the department's transplant database and medical records, and the donor's data was retrieved from the DonorNet. The data included demographic data, lung heights, measured total lung capacity (TLC) from plethysmography for recipients and estimated TLC for donors, clinical data, and CT-derived lung volumes in both pre- and post-transplant recipients. The post-transplant CT-derived lung volume in recipients was used as a surrogate for donor lung CT volumes due to inadequate or poor donor CT data. Computed tomography-derived lung volumes were calculated using thresholding, region growing, and cutting techniques on Computer-Aided Design and Mimics (Materialise NV, Leuven, Belgium) programs. Preoperative CT-derived lung volumes in recipients were compared with the plethysmography TLC, Frustum Model, and donor-predicted TLC. The ratio of the recipient's pre-and postoperative CT-derived volumes, the ratio of preoperative CT-derived lung volume, and donor-estimated TLC were studied to detect a correlation with 1-year outcomes. RESULTS: The recipient preoperative CT-derived volume correlated with the recipient preoperative plethysmography TLC (Pearson correlation coefficient [PCC] of 0.688) and with the recipient Frustum model volume (PCC of 0.593). The recipient postoperative CT-derived volume correlated with the recipient's postoperative plethysmography TLC (PCC of 0.651). There was no statistically significant correlation between recipients' CT-derived pre- or postoperative volume with donor-estimated TLC. The ratio of preoperative CT-derived volume to donor-estimated TLC correlated inversely with the length of ventilation (P value = .0031). The ratio of postoperative CT-derived volume to preoperative CT-derived volume correlated inversely with delayed sternal closure (P = .0039). No statistically significant correlations were found in evaluating outcomes related to lung oversizing in the recipient (defined as a postoperative to preoperative CT-derived lung volume ratio of >1.2). CONCLUSIONS: Generating CT-derived lung volumes is a valid and convenient method for evaluating lung volumes for transplantation in patients with ILD and/or IPF. Donor-estimated TLC should be interpreted carefully. Further studies should derive donor lung volumes from CT scans for a more accurate evaluation of lung size matching.

Bispecific antibody CAP256.J3LS targets V2-apex and CD4-binding sites with high breadth and potency.

Antibody CAP256-VRC26.25 targets the second hypervariable region (V2) at the apex of the HIV envelope (Env) trimer with extraordinary neutralization potency, although less than optimal breadth. To improve breadth, we linked the light chain of CAP256V2LS, an optimized version of CAP256-VRC26.25 currently under clinical evaluation, to the llama nanobody J3, which has broad CD4-binding site-directed neutralization. The J3-linked bispecific antibody exhibited improved breadth and potency over both J3 and CAP256V2LS, indicative of synergistic neutralization. The cryo-EM structure of the bispecific antibody in complex with a prefusion-closed Env trimer revealed simultaneous binding of J3 and CAP256V2LS. We further optimized the pharmacokinetics of the bispecific antibody by reducing the net positive charge of J3. The optimized bispecific antibody, which we named CAP256.J3LS, had a half-life similar to CAP256V2LS in human FcRn knock-in mice and exhibited suitable auto-reactivity, manufacturability, and biophysical risk. CAP256.J3LS neutralized over 97% of a multiclade 208-strain panel (geometric mean concentration for 80% inhibition (IC80) 0.079 µg/ml) and 100% of a 100-virus clade C panel (geometric mean IC80 of 0.05 µg/ml), suggesting its anti-HIV utility especially in regions where clade C dominates.

Anteromedial Tibial Attachment in Single-Bundle Anterior Cruciate Ligament Reconstruction Can Represent Normal Kinematics in Computer Simulation.

Tunnel position during anterior cruciate ligament (ACL) reconstruction is considered as an important factor to restore normal knee kinematics and to gain better clinical outcomes. It is still unknown where the optimal femoral and tibial tunnel position is located in single-bundle (SB) ACL reconstruction. The purposes of this study were to analyze the knee kinematics with various graft positions and to propose the optimal graft position during SB ACL reconstruction. A musculoskeletal computer simulation was used to analyze knee kinematics. Four attachments on the femoral side (anteromedial [AM], mid, posterolateral [PL], and over-the-top positions) and three attachments on the tibial side (AM, middle, and PL positions) were determined. The middle-bundle attachment was placed at the midpoint of the AM and PL bundle attachments for the femoral and tibial attachments. SB ACL reconstruction models were constructed to combine each of the four femoral attachments with each of three tibial attachments. Kinematic comparison was made among a double-bundle (DB) model and 12 SB reconstruction models during deep knee bend and stair descent activity. The tunnel position of the tibia had greater effect of knee kinematics than that of the femur. AM tibial attachment models showed similar medial and lateral anteroposterior positions to the DB model for both activities. Axial rotation in the AM tibial attachment models was similar to the DB model regardless of the femoral attachment, whereas greater maximum axial rotation was exhibited in the PL tibial attachment models, especially during stair descent activity. AM tibial attachment can represent normal knee kinematics, whereas the PL tibial attachment can induce residual rotational instability during high-demand activities. The AM tibial tunnel is recommended for SB ACL reconstruction.

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.

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.

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.

Lucid Dreams Associated with Pregabalin: Implications for Clinical Practice.

Here, we describe a case of a patient with multiple myeloma who reported symptoms of lucid dreams. This patient was taking methadone for neoplasm related pain. The patient was also taking pregabalin which was initially started at dosing of 50 mg taken orally three times a day. Five days after initiation of pregabalin, the dosing was increased to 100 mg taken orally three times daily. The patient developed lucid dreams during a hospital stay and methadone was initially thought to be the cause of her lucid dreams. Methadone dosing was decreased with no success, and her lucid dreams persisted. On the patient's thirteenth day of hospital admission, the pregabalin dose was decreased from 100 mg three times a day to 75 mg twice daily. Five days later, the pregabalin was decreased from 75 mg twice daily to 50 mg twice daily. Pregabalin was continued for five more days and then discontinued. Resolution of the lucid dreams occurred following discontinuation of pregabalin. To our knowledge, this is the first reported case of an association between pregabalin and lucid dreams. Given that pregabalin is a widely used medication, we found this case to be relevant to describe this unique presentation.

Structural basis for llama nanobody recognition and neutralization of HIV-1 at the CD4-binding site.

Nanobodies can achieve remarkable neutralization of genetically diverse pathogens, including HIV-1. To gain insight into their recognition, we determined crystal structures of four llama nanobodies (J3, A12, C8, and D7), all of which targeted the CD4-binding site, in complex with the HIV-1 envelope (Env) gp120 core, and determined a cryoelectron microscopy (cryo-EM) structure of J3 with the Env trimer. Crystal and cryo-EM structures of J3 complexes revealed this nanobody to mimic binding to the prefusion-closed trimer for the primary site of CD4 recognition as well as a secondary quaternary site. In contrast, crystal structures of A12, C8, and D7 with gp120 revealed epitopes that included portions of the gp120 inner domain, inaccessible on the prefusion-closed trimer. Overall, these structures explain the broad and potent neutralization of J3 and limited neutralization of A12, C8, and D7, which utilized binding modes incompatible with the neutralization-targeted prefusion-closed conformation of Env.

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.

Rate of cannabis use in older adults with cancer.

OBJECTIVES: Older adults with cancer are increasingly inquiring about and using cannabis. Despite this, few studies have examined cannabis use in patients with cancer aged 65 years and older as a separate group and identified characteristics associated with use. The current study sought to determine the rate of cannabis use in older adult patients with cancer and to identify demographic and clinical correlates of use. METHODS: We conducted a retrospective review of patients with cancer referred for specialised symptom management between January 2014 and May 2017 who underwent routine urine drug testing for tetrahydrocannabinol as part of their initial clinic visit. RESULTS: Approximately 8% (n=24) of patients with cancer aged 65 years and older tested positive for tetrahydrocannabinol compared with 30% (n=51) of young adults and 21% (n=154) of adults. At the univariate level, more cannabis users had lower performance status than non-users (p=0.02, Fisher's exact test). There were no other demographic and clinical characteristics significantly associated with cannabis use in older adults. CONCLUSIONS: Older adult patients made up nearly 25% (n=301) of the total sample and had a rate of cannabis use of 8%. As one of the first studies to assess cannabis use via objective testing rather than self-report, this study adds significantly to the emerging literature on cannabis use in people aged 65 years and older. Findings suggest the rate of use in older adults living with cancer is higher than that among older adults in the general population.

Subcutaneous and visceral adipose tissue in patients with primary and recurrent incisional hernia.

PURPOSE: Visceral obesity rather than body mass index has been reported to be associated with a higher incidence of incisional hernias. The aim of this study was to examine the relationship between CT measured adipose tissue and muscle in primary and recurrent incisional hernia. METHODS: Patients with a 'Primary' or 'Recurrent incisional hernia' were obtained from a prospective cohort of patients who were being assessed for incisional hernia repair over a 2-year period. Computerised tomography (CT)-images were analysed using NIH Image-J software to quantify adipose tissue and skeletal muscle cross-sectional areas at the level of lumber vertebra 3/4 using standard Hounsfield units. To test inter-observer 'absolute agreement', each parameter was measured independently by two investigators and reliability analysis performed. RESULTS: Thirty-six patients were included in the study: 15 had a Primary while 21 had a Recurrent incisional hernia. Both groups had similar baseline characteristics. Reliability analysis for CT-measured areas showed very high interclass correlation coefficient (ICC) between observers. Patients in the recurrent group had significantly greater subcutaneous adipose tissue (SAT) [median = 321.9cm2 vs 230.9cm2, p = 0.04] and visceral adipose tissue (VAT) [median = 221.1cm2 vs 146.8cm2, p = 0.03] than those in the primary group. There was no difference in skeletal muscle areas for right [median = 2.8cm2 vs 2.9cm2] and left [median = 3.7cm2 vs 4.1cm2] rectus muscles between groups. CONCLUSION: Our study shows that patients with a recurrent incisional hernia have significantly more subcutaneous and visceral adipose tissue than those with a primary incisional hernia. Further studies in this area are required if we are to reduce the burden of recurrent hernia following repair of a primary incisional hernia.